Scaffolds with viable tissue

ABSTRACT

A composite implant is provided for repairing a tissue defect in a patient. In one embodiment, the implant is a porous tissue scaffold having at least one pocket formed therein and adapted to contain a viable tissue. The tissue scaffold can have a variety of configurations, and in one embodiment it includes top and bottom portions that can be at least partially mated to one another, and in an exemplary embodiment that are heated sealed to one another around a perimeter thereof to form an enclosed pocket therebetween. The pocket is preferably sealed with a viable tissue disposed therein. In another embodiment, the tissue scaffold is substantially wedge-shaped and the pocket comprises a hollow interior formed in the tissue scaffold, and/or at least one lumen extending into the tissue scaffold. The tissue scaffold can also optionally include at least one surface feature formed thereof to promote blood vessel formation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/775,034 entitled “Scaffolds With Viable Tissue” filed Feb.9, 2004, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and devices for repairing andreplacing torn or damaged tissue, and in particular to tissue implantshaving viable tissue capable of tissue regeneration and integration withtissue surrounding the area to be repaired, as well as methods for usingsuch tissue implants.

BACKGROUND OF THE INVENTION

Injuries to tissue, such as cartilage, skin, muscle, bone, tendon, andligament where the tissue has been injured or traumatized frequentlyrequire surgical intervention to repair the damage and facilitatehealing. Such surgical repairs can include suturing or otherwiserepairing the damaged tissue with known medical devices, augmenting thedamaged tissue with other tissue, using an implant, a graft or anycombination of these techniques. Despite these conventional methods oftissue repair, there continues to be a need for surgical solutions thatfacilitate the regeneration of new, healthy tissue to provide morereliable repair and healing of the injured or damaged tissue over thelong term.

The search for a reliable source of viable cells for tissue regenerationhas been pursued for years. Recent tissue engineering techniques forrepairing tissue have typically involved replacing or reconstructingdamaged or injured tissue with cells that have been manipulated ex vivoto stimulate new tissue growth. The cells are usually incorporated intoa delivery vehicle (e.g., a scaffold or surgical implant) for placementat the tissue site, whereupon new tissue can be grown. Various surgicalimplants are known and have been used in surgical procedures to helpachieve these benefits. For example, it is known to use various devicesand techniques for creating implants having isolated cells loaded onto adelivery vehicle. Such cell-seeded implants have been used in an invitro method of making and/or repairing cartilage by growingcartilaginous structures that consist of chondrocytes seeded ontobiodegradable, biocompatible fibrous polymeric matrices as well asmatrices developed from collageneous materials. Such methods require theinitial isolation of chondrocytes from cartilaginous tissue prior to thechondrocytes being seeded onto the polymeric matrices. Other techniquesfor repairing damaged tissue employ implants having stem or progenitorcells that are used to produce the desired tissue. For example, it isknown to use stem or progenitor cells, such as the cells within fattytissue, muscle, bone marrow, or embryonic tissue to regenerate bone,cartilage, and other soft tissues in a patient. For example, stem cellsfrom fat are removed from the patient and placed in an environmentfavorable to cartilage formation, thereby inducing the cells toproliferate and to create a different type of cell, such as cartilagecells.

While the trend towards using tissue engineering approaches to tissuerepair continues to gain popularity, mainly because of the long-termbenefits provided to the patient, these current techniques are notwithout drawbacks. One disadvantage with current tissue engineeringtechniques is that they can be time consuming. A typical processinvolves the harvest of a tissue sample from the patient in a firstsurgical procedure, which is then transported to a laboratory for cellisolation, culture and amplification. The cell sample is grown for aperiod of 3 to 4 weeks using standard cell culture techniques to createa cell bank. Once the cell population has reached a target number, thecells are sent back to the surgeon for implantation during a secondsurgical procedure. This manual, labor-intensive process is extremelycostly and time consuming. Although the clinical data suggest long-termbenefits for the patient, the prohibitive cost of the procedure,combined with the traumatic impact of two surgical procedures, hashampered adoption of these techniques. And though allografts have beenused for tissue repair in the past, this solution is also not idealbecause of the limited availability of graft material and the potentialfor disease transmission.

For these reasons, there continues to exist a need in this art for noveldevices and methods for regenerating tissue which are less timeconsuming and easier to implement. It is also desirable to provide animplant which can serve as a reliable source of viable cells, and whichcan be made in a quick and efficient manner for immediate use duringsurgery. There is thus a need for a less costly solution to repairingtissue defects or injuries that also provides the advantages of tissueregeneration, without the encumbrances of the currently availabledevices and methods of tissue repair previously mentioned.

SUMMARY OF THE INVENTION

The present invention generally provides a composite implant forrepairing a tissue defect in a patient. In one embodiment, the implantis a porous tissue scaffold having at least one pocket formed thereinand adapted to contain a viable tissue. The tissue scaffold can have avariety of shapes and configurations, and in one embodiment it includestop and bottom portions. The top and bottom portions can be at leastpartially mated to one another, and in an exemplary embodiment they areheat sealed to one another around a perimeter thereof to form anenclosed pocket therebetween. Heat sealing can be performed eitherbefore or after the viable tissue is loaded into the scaffold. Inanother embodiment, the tissue scaffold is substantially wedge-shapedand the pocket comprises a hollow interior formed in the tissuescaffold, and/or at least one lumen extending into the tissue scaffold.The tissue scaffold can also optionally include at least one surfacefeature formed thereon to promote blood vessel formation. By way ofnon-limiting example, the surface feature can be in the form of aplurality of channels formed on an outer surface of the tissue scaffold.

In a further embodiment of the present invention, the tissue scaffoldcan include a viable tissue disposed within the pocket. The viabletissue should be effective to migrate into the scaffold to integratewith native tissue surrounding the scaffold. At least one bioactivesubstance can be applied to the viable tissue to stimulate cell growth.Suitable bioactive substances include, for example, blood clots,platelet rich plasma, cartilage-derived morphogenic proteins,recombinant human growth factors, and combinations thereof. In anotherembodiment, the bioactive substance can additionally or alternatively beapplied to the tissue scaffold.

The present invention also provides a method for repairing defectivetissue that includes the steps of providing a tissue scaffold having atleast one pocket formed therein and adapted to contain a viable tissue,obtaining a viable tissue, loading the viable tissue into the at leastone pocket of the tissue scaffold, and implanting the tissue scaffoldwith the viable tissue disposed therein at a defect site in a patient'sbody. The method can also include the step of applying at least onebioactive substance to the viable tissue to stimulate cell growth.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is a perspective view of one embodiment of a composite implant inaccordance with the present invention;

FIG. 2 is a perspective view of another embodiment of a compositeimplant in accordance with the present invention;

FIG. 3 is a perspective view of a composite implant in accordance withanother embodiment the present invention;

FIG. 4 is a perspective view of yet another embodiment of a compositeimplant in accordance with the present invention;

FIG. 5A is an illustration of a composite implant having a top portionand a bottom portion with a viable tissue disposed thereon in accordancewith another embodiment of the present invention;

FIG. 5B is an illustration of a heat sealing apparatus in accordancewith the present invention;

FIG. 5C illustrates the composite implant of FIG. 5A after being sealedusing the heat sealing apparatus of FIG. 5B;

FIG. 6 is a perspective view of yet another embodiment of a compositeimplant having surface features formed thereon according to anotherembodiment of the present invention;

FIG. 7 illustrates another embodiment of a composite implant havingsurface features formed thereon;

FIG. 8A is a photomicrograph of a small intestine submucosa (SIS)composite implant serving as a control;

FIG. 8B is a photomicrograph of an SIS composite implant having a pocketcontaining a viable tissue formed from bovine minced meniscal tissue(BMT), showing native meniscal tissue cells migrating into the implant;

FIG. 8C is a photomicrograph of an SIS composite implant having a pocketcontaining a viable tissue formed from BMT and platelet rich plasma(PRP), showing improved native tissue cell migration and integrationinto the implant;

FIG. 8D is a photomicrograph of an SIS composite implant, having apocket containing a viable tissue source that was prepared by combiningthe BMT and PRP with a bioresorbable polymer scaffold (referred to as“FPV”) in a 50:50 ratio, showing improved native meniscal tissue cellmigration and integration into the implant;

FIG. 8E is a photomicrograph of an SIS composite implant, having apocket containing a viable tissue source that was prepared by combiningthe BCT and PRP with a bioresorbable polymer scaffold (referred to as“PV”) in a 50:50 ratio, showing improved native tissue cell migrationand integration into the implant;

FIG. 9A is a bar chart of viable meniscal tissue cell migration intoimplants formed from 65% polyglycolic acid/35% polycaprolactone andreinforced with a PDS® mesh, comparing an implant having no bioactivesubstance to an implant treated with i blood, an implant treated withPRP, and an implant treated with eight times concentrated PRP;

FIG. 9B is a bar chart comparing viable tissue cell migration intoimplants formed from 65% polyglycolic acid/35% polycaprolactone andreinforced with a PDS® mesh, comparing implants containing viable tissuetreated with varying amounts of cartilage-derived morphogenic proteins(CDMP-1);

FIG. 10A is a photomicrograph of an implant formed from minced meniscaltissue serving as a control; and

FIG. 10B is a photomicrograph of an implant formed from minced meniscaltissue loaded with CDMP-1, showing native tissue cell migration into theimplant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides a composite implant thatincludes a tissue scaffold having at least one pocket formed therein,and a viable tissue disposed within the pocket of the tissue scaffold.The use of a pocket is particularly advantageous in that it helps retainviable tissue within the composite implant and it minimizes the loss ofviable tissue following placement at a defect site in a patient. Thepocket is also advantageous in that the viable tissue can migrate andpopulate the scaffold, thereby enhancing the integration between thenative tissue and the composite implant.

A person skilled in the art will appreciate that the biocompatiblecomposite implants of the present invention can be used in the treatmentof various types of tissue for various purposes, including but notlimited to tissue repair, tissue bulking, cosmetic treatments,therapeutic treatments, tissue remodeling or augmentation, and tissuesealing. In an exemplary embodiment, the implants are used for therepair and/or regeneration of diseased or damaged tissue.

The tissue scaffold used to form the composite implant of the presentinvention can have various configurations, shapes, and sizes. FIGS. 1-7illustrate a variety of exemplary composite implants, each formed from atissue scaffold having at least one pocket formed therein containing aviable tissue. As stated above, the pocket allows the viable tissue togrow into and through the scaffold, such that the tissue becomesembedded in and integrated with the scaffold.

In FIGS. 1 and 2, the composite implant 10, 20 is formed from a tissuescaffold 12, 14 that is substantially wedge-shaped, and in particularthat includes opposed top and bottom walls 12 a, 12 b, 22 a, 22 b,opposed side walls 12 c, 12 d, 22 c, 22 d extending between the top andbottom walls 12 a, 12 b, 22 a, 22 b, and an end wall 12 e, 22 e thatconnects the top, bottom, and opposed side walls 12 a, 12 b, 12 c, 12 d,22 a, 22 b, 22 c, 22 d. The pocket 14, 24 in each scaffold 12, 22 isformed as a lumen that extends into the end wall 12 e, 22 e of eachscaffold 12, 22. While the opening of the pocket 14, 24 can be formed inany wall of the implant 10, 20, an end wall 12 e, 22 e opening isparticularly advantageous in that it simplifies loading of the implantwith the viable tissue 16, 26. Moreover, when the composite implant 10,20 is implanted, native tissue surrounding the scaffold 12, 22 will abutthe pocket opening to help maintain the viable tissue 16, 26 therein.

Still referring to FIGS. 1 and 2, while the pocket 14, 24 in eachimplant 10, 20 can have virtually any shape and size, the opening of thepocket 14 shown in FIG. 1 is substantially rectangular in shape, and theopening of the pocket 24 shown in FIG. 2 is in the form of a cruciform.Each pocket is preferably substantially centrally located in the endwall 12 e, 22 e of the scaffold 12, 22, as shown, and each pocketpreferably extends through a substantial portion of the scaffold 12, 22.This construction facilitates migration of the viable tissue through theentire scaffold 12, 22 in all directions.

FIG. 3 illustrates another embodiment of a composite implant 30. In thisembodiment, the tissue scaffold 32 includes upper and lower portions orlayers 32 a, 32 b that are configured to sandwich a viable tissue 36 ina pocket 34 formed therebetween. The viable tissue 36 can merely beplaced on one or the layers, e.g., lower layer 32 b, and the upper layer32 a can then be positioned thereon to form the composite implant. Aperson skilled in the art will appreciate that while the illustratedlayers 32 a, 32 b are substantially rectangular, each layer can havevirtually any shape and size.

FIG. 4 illustrates yet another embodiment of a composite implant 40having a tissue scaffold 42 that includes upper and lower layers 42 a,42 b with a viable tissue 46 disposed in a pocket 44 formedtherebetween. The upper and lower layers 42 a, 42 b are mated to oneanother at only one end thereof. Thus, while the scaffold 42 issubstantially wedge shaped, it does not include sidewalls that are matedto one another as shown in FIGS. 1 and 2.

In another embodiment of the present invention, one or more edges of thetissue scaffold can be at least partially sealed to one another to formthe pocket which is adapted to contain a viable tissue. By way ofnon-limiting example, FIGS. 5A and 5C illustrate a composite implant 50formed from a tissue scaffold 52 having top and bottom layers 52 a, 52 bthat are sealed to one another around a perimeter thereof to retain aviable tissue 56 disposed therebetween. While virtually any techniquecan be used to mate the layers 52 a, 52 b to one another, in anexemplary embodiment the layers 52 a, 52 b are heat sealed to oneanother. Heat sealing can be performed before or after the viable tissue56 is placed between the layers 52 a, 52 b, however care should be takento avoid damage to the viable tissue 56. By way of non-limiting example,FIG. 5B illustrates a heat sealing apparatus 100 that includes a lowermember or tray 102 b that is effective to seat the layers 52 a, 52 bwith the viable tissue 56 disposed therebetween. The heat sealingapparatus 100 also includes an upper member 102 a that is preferablyconnected to the lower member 102 b by a lever or hinge 104, and that iseffective to apply heat to the perimeter of the tissue scaffold 52 toseal the layers 52 a, 52 b to one another to form the composite implant50 shown in FIG. 5C. In use, the heat sealed scaffold is particularlyadvantageous in that it prevents the viable tissue from migrating fromthe scaffold, and it can also be used to provide mechanicalreinforcement to the implant for receiving a suture or other attachmentmechanism for securing the implant to surrounding tissue. A personskilled in the art will appreciate that a variety of techniques can beused to mate the layers to one another including, for example, adhesive,sutures, a fold or roll seal, a mechanical press seal, etc.

In other embodiments of the present invention, the composite implant caninclude one or more surface features formed on the tissue scaffold topromote and/or guide blood vessel formation. FIGS. 6 and 7 illustrateexemplary implants 60, 70 with surface features formed thereon. Asshown, each implant 60, 70 generally includes a tissue scaffold 62, 72formed from top and bottom portions 62 a, 62 b, 72 a, 72 b that at matedto one another at one end thereof such that each implant 60, 70 issubstantially wedge-shaped. A viable tissue 66, 76 is disposed withinthe pocket 64, 74 that is formed between the top and bottom portions 62a, 62 b, 72 a, 72 b. In the embodiment illustrated in FIG. 6, thesurface features are formed on the top and bottom layers 62 a, 62 b, andthey are in the form of channels 68 that extend there across and thatare spaced apart from one another. In the embodiment illustrated in FIG.7, the surface features are also formed on the top and bottom layers 72a, 72 b, however they are in the form of bores 78 formed therein. Aperson skilled in the art will appreciate that the location and quantityof surface features can vary.

The materials used to form the composite implant of the presentinvention can also vary, and a variety of materials and techniques forforming a tissue scaffold are known in the art and can be used with thepresent invention. In an exemplary embodiment, however, the tissuescaffold is formed using a material or delivery vehicle that isbiocompatible and that has sufficient structural integrity and physicaland/or mechanical properties to effectively provide for ease of handlingin an operating room environment. Sufficient strength and physicalproperties can be developed in the scaffold through the selection ofmaterials used to form the scaffold, and the manufacturing process. Inaddition, the scaffold is preferably sufficiently porous to allow cellgrowth therein. Preferably, the median pore size is in the range ofabout 100 to 500 microns. The scaffold can also optionally besufficiently pliable to allow the scaffold to adjust to the dimensionsof the target site of implantation, and/or to accommodate tissue growthwithin the interior region of the scaffold, so that the geometry of thescaffold can be remodeled as tissue ingrowth increases.

In an exemplary embodiment, the scaffold is formed from a bioresorbableor bioabsorbable material, and more preferably from a bioresorbable orbioabsorbable material that has the ability to resorb in a timelyfashion in the body environment. The differences in the absorption timeunder in vivo conditions can also be the basis for combining twodifferent copolymers when forming the scaffolds of the presentinvention. For example, a copolymer of 35:65 ε-caprolactone andglycolide (a relatively fast absorbing polymer) can be blended with40:60 ε-caprolactone and L-lactide copolymer (a relatively slowabsorbing polymer) to form a biocompatible scaffold. Depending upon theprocessing technique used, the two constituents can be either randomlyinter-connected bicontinuous phases, or the constituents could have agradient-like architecture in the form of a laminate-type composite witha well integrated interface between the two constituent layers. Themicrostructure of these scaffolds can be optimized to regenerate orrepair the desired anatomical features of the tissue that is beingregrown.

In one embodiment of the present invention, the scaffold can be formedfrom a biocompatible polymer. A variety of biocompatible polymers can beused to make the biocompatible tissue implants or scaffold devicesaccording to the present invention. The biocompatible polymers can besynthetic polymers, natural polymers or combinations thereof. As usedherein the term “synthetic polymer” refers to polymers that are notfound in nature, even if the polymers are made from naturally occurringbiomaterials. The term “natural polymer” refers to polymers that arenaturally occurring.

In embodiments where the scaffold includes at least one syntheticpolymer, suitable biocompatible synthetic polymers can include polymersselected from the group consisting of aliphatic polyesters, poly(aminoacids), copoly(ether-esters), polyalkylenes oxalates, polyamides,tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, poly(propylene fumarate),polyurethane, poly(ester urethane), poly(ether urethane), and blends andcopolymers thereof. Suitable synthetic polymers for use in the presentinvention can also include biosynthetic polymers based on sequencesfound in collagen, laminin, glycosaminoglycans, elastin, thrombin,fibronectin, starches, poly(amino acid), gelatin, alginate, pectin,fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronicacid, silk, ribonucleic acids, deoxyribonucleic acids, polypeptides,proteins, polysaccharides, polynucleotides and combinations thereof.

For the purpose of this invention aliphatic polyesters include, but arenot limited to, homopolymers and copolymers of lactide (which includeslactic acid, D-,L- and meso lactide); glycolide (including glycolicacid); ε-caprolactone; p-dioxanone (1,4-dioxan-2-one); trimethylenecarbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylenecarbonate; δ-valerolactone; β-butyrolactone; γ-butyrolactone;α-decalactone; hydroxybutyrate; hydroxyvalerate; 1,4-dioxepan-2-one(including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione);1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine;pivalolactone; α,αdiethylpropiolactone; ethylene carbonate; ethyleneoxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,6-dimethyl-dioxepan-2-one;6,8-dioxabicycloctane-7-one and polymer blends thereof. Aliphaticpolyesters used in the present invention can be homopolymers orcopolymers (random, block, segmented, tapered blocks, graft, triblock,etc.) having a linear, branched or star structure. Other useful polymersinclude polyphosphazenes, co-, ter- and higher order mixed monomer basedpolymers made from L-lactide, D,L-lactide, lactic acid, glycolide,glycolic acid, para-dioxanone, trimethylene carbonate andε-caprolactone.

In embodiments where the scaffold includes at least one natural polymer,suitable examples of natural polymers include, but are not limited to,fibrin-based materials, collagen-based materials, hyaluronic acid-basedmaterials, glycoprotein-based materials, cellulose-based materials,silks and combinations thereof. By way of non-limiting example, thebiocompatible scaffold can be constructed from a collagen-based smallintestine submucosa.

In still yet another embodiment, the preferred scaffold for tissuerepair, including cartilage, meniscus, tendon, ligament, and skinrepair, is constructed from a naturally occurring extracellular matrixmaterial (“ECM”), such as that found in the stomach, bladder,alimentary, respiratory, urinary, integumentary, genital tracts, orliver basement membrane of animals. Preferably, the ECM is derived fromthe alimentary tract of mammals, such as cows, sheeps, dogs, cats, andmost preferably from the intestinal tract of pigs. The ECM is preferablysmall intestine submucosa (“SIS”), which can include the tunicasubmucosa, along with basilar portions of the tunica mucosa,particularly the lamina muscularis mucosa and the stratum compactum.

For the purposes of this invention, it is within the definition of anaturally occurring ECM to clean and/or comminute the ECM, or even tocross-link the collagen fibers within the ECM. Also, while reference ismade to SIS, it is understood that other naturally occurring ECMs arewithin the scope of this invention. Thus, as used herein, the terms“naturally occurring extracellular matrix” or “naturally occurring ECM”can refer to extracellular matrix material that has been cleaned,disinfected, sterilized, and optionally cross-linked.

Where SIS is used, an SIS graft can be harvested in a variety of ways,as will be understood by one skilled in the art. The resulting graftmaterial can have a variety of geometries and consistencies includingfor example, coiled, helical, spring-like, randomized, branched,sheet-like, tubular, spherical, fragmented, fluidized, comminuted,liquefied, foamed, suspended, gel-like, injectable, powdered, ground,and sheared.

In yet another embodiment of the present invention, the scaffold can beformed using tissue grafts, such as may be obtained from autogeneictissue, allogeneic tissue and xenogeneic tissue. By way of non-limitingexample, tissues such as skin, cartilage, ligament, tendon, periosteum,perichondrium, synovium, fascia, mesenter and sinew can be used astissue grafts to form the biocompatible scaffold. In some embodimentswhere an allogeneic tissue is used, tissue from a fetus or newborns canbe used to avoid the immunogenicity associated with some adult tissues.

In other embodiments of the present invention, the tissue scaffold canbe formed from elastomeric copolymers such as, for example, polymershaving an inherent viscosity in the range of about 1.2 dL/g to 4 dL/g,more preferably about 1.2 dL/g to 2 dL/g, and most preferably about 1.4dL/g to 2 dL/g as determined at 25° C. in a 0.1 gram per deciliter(g/dL) solution of polymer in hexafluoroisopropanol (HFIP). Suitableelastomers also preferably exhibit a high percent elongation and a lowmodulus, while possessing good tensile strength and good recoverycharacteristics. In the preferred embodiments of this invention, theelastomer exhibits a percent elongation greater than about 200 percentand preferably greater than about 500 percent. In addition to theseelongation and modulus properties, the elastomers should also have atensile strength greater than about 500 psi, preferably greater thanabout 1,000 psi, and a tear strength of greater than about 50 lbs/inch,preferably greater than about 80 lbs/inch.

Exemplary biocompatible elastomers include, but are not limited to,elastomeric copolymers of ε-caprolactone and glycolide with a mole ratioof ε-caprolactone to glycolide of from about 35:65 to about 65:35, morepreferably from 45:55 to 35:65; elastomeric copolymers of ε-caprolactoneand lactide (including L-lactide, D-lactide, blends thereof, and lacticacid polymers and copolymers) where the mole ratio of ε-caprolactone tolactide is from about 95:5 to about 30:70 and more preferably from 45:55to 30:70 or from about 95:5 to about 85:15; elastomeric copolymers ofp-dioxanone (1,4-dioxan-2-one) and lactide (including L-lactide,D-lactide, blends thereof, and lactic acid polymers and copolymers)where the mole ratio of p-dioxanone to lactide is from about 40:60 toabout 60:40; elastomeric copolymers of ε-caprolactone and p-dioxanonewhere the mole ratio of ε-caprolactone to p-dioxanone is from about from30:70 to about 70:30; elastomeric copolymers of p-dioxanone andtrimethylene carbonate where the mole ratio of p-dioxanone totrimethylene carbonate is from about 30:70 to about 70:30; elastomericcopolymers of trimethylene carbonate and glycolide (includingpolyglycolic acid) where the mole ratio of trimethylene carbonate toglycolide is from about 30:70 to about 70:30; elastomeric copolymers oftrimethylene carbonate and lactide (including L-lactide, D-lactide,blends thereof, and lactic acid polymers and copolymers) where the moleratio of trimethylene carbonate to lactide is from about 30:70 to about70:30; and blends thereof. Other examples of suitable biocompatibleelastomers are described in U.S. Pat. No. 5,468,253.

In another embodiment of the present invention, the tissue scaffold canbe formed from an elastomer that is a copolymer of 35:65 ε-caprolactoneand glycolide, formed in a dioxane solvent and including a polydioxanonemesh. In another embodiment, the elastomer used to form the tissuescaffold can be a copolymer of 40:60 ε-caprolactone and lactide with apolydioxanone mesh. In yet another embodiment, the elastomer is a 50:50blend of a 35:65 copolymer of ε-caprolactone and glycolide and 40:60copolymer of ε-caprolactone and lactide. The polydioxanone mesh may bein the form of a one layer thick two-dimensional mesh or a multi-layerthick three-dimensional mesh.

In another embodiment of the present invention, the tissue scaffold canbe formed from a biocompatible ceramic material. Suitable biocompatibleceramic materials include, for example, hydroxyapatite, α-tricalciumphosphate, β-tricalcium phosphate, bioactive glass, calcium phosphate,calcium sulfate, calcium carbonate, xenogeneic and allogeneic bonematerial and combinations thereof. Suitable bioactive glass materialsfor use in the present invention include silicates containing calciumphosphate glass, or calcium phosphate glass with varying amounts ofsolid particles added to control resorption time. Suitable compoundsthat may be incorporated into the calcium phosphate bioactive glassinclude, but are not limited to, magnesium oxide, sodium oxide,potassium oxide, and combinations thereof.

In yet another embodiment of the present invention, the scaffold can beformed from a polymeric foam component having pores with an open cellpore structure. The pore size can vary, but preferably, the pores aresized to allow tissue ingrowth. More preferably, the pore size is in therange of about 50 to 1000 microns, and even more preferably, in therange of about 50 to 500 microns. The polymeric foam component can,optionally, contain a reinforcing component, such as for example, thetextiles disclosed above. In some embodiments where the polymeric foamcomponent contains a reinforcing component, the foam component can beintegrated with the reinforcing component such that the pores of thefoam component penetrate the mesh of the reinforcing component andinterlock with the reinforcing component.

It may also be desirable to use polymer blends to form scaffolds whichtransition from one composition to another composition in agradient-like architecture. Scaffolds having this gradient-likearchitecture are particularly advantageous in tissue engineeringapplications to repair or regenerate the structure of naturallyoccurring tissue such as cartilage (articular, meniscal, septal,tracheal, auricular, costal, etc.), tendon, ligament, nerve, esophagus,skin, bone, and vascular tissue. For example, by blending an elastomerof α-caprolactone-co-glycolide with E-caprolactone-co-lactide (e.g.,with a mole ratio of about 5:95) a scaffold may be formed thattransitions from a softer spongy material to a stiffer more rigidmaterial, for example, in a manner similar to the transition fromcartilage to bone. Clearly, one skilled in the art will appreciate thatother polymer blends may be used for similar gradient effects, or toprovide different gradients (e.g., different absorption profiles, stressresponse profiles, or different degrees of elasticity).

One of ordinary skill in the art will appreciate that the selection of asuitable material for forming the biocompatible scaffold of the presentinvention depends on several factors. These factors include in vivomechanical performance; cell response to the material in terms of cellattachment, proliferation, migration and differentiation;biocompatibility; and optionally, bioabsorption (or biodegradation)kinetics. Other relevant factors include the chemical composition,spatial distribution of the constituents, the molecular weight of thepolymer, and the degree of crystallinity.

The tissue scaffold used to form the composite implant can also includea reinforcing material comprised of any absorbable or non-absorbabletextile having, for example, woven, knitted, warped knitted (i.e.,lace-like), non-woven, and braided structures. In one embodiment, thereinforcing material has a mesh-like structure. In any of the abovestructures, mechanical properties of the material can be altered bychanging the density or texture of the material, the type of knit orweave of the material, the thickness of the material, or by embeddingparticles in the material. The mechanical properties of the material mayalso be altered by creating sites within the mesh where the fibers arephysically bonded with each other or physically bonded with anotheragent, such as, for example, an adhesive or a polymer.

The fibers used to make the reinforcing component can includemonofilaments, yarns, threads, braids, or bundles of fibers. Thesefibers can be made of any biocompatible material including bioabsorbablematerials such as polylactic acid (PLA), polyglycolic acid (PGA),polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate(TMC), copolymers or blends thereof. These fibers can also be made fromany biocompatible materials based on natural polymers including silk andcollagen-based materials. These fibers can also be made of anybiocompatible fiber that is nonresorbable, such as, for example,polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene),polycarbonate, polypropylene and poly(vinyl alcohol). In one embodiment,the fibers are formed from 95:5 copolymer of lactide and glycolide.

In another embodiment, the fibers that form the reinforcing material canbe made of a bioabsorbable glass. Bioglass, a silicate containingcalcium phosphate glass, or calcium phosphate glass with varying amountsof solid particles added to control resorption time are examples ofmaterials that could be spun into glass fibers and used for thereinforcing material. Suitable solid particles that may be added includeiron, magnesium, sodium, potassium, and combinations thereof.

The biocompatible scaffolds as well as the reinforcing material may alsobe formed from a thin, perforation-containing elastomeric sheet withpores or perforations to allow tissue ingrowth. Such a sheet could bemade of blends or copolymers of polylactic acid (PLA), polyglycolic acid(PGA), polycaprolactone (PCL), and polydioxanone (PDO).

A person skilled in the art will appreciate that one or more layers ofthe reinforcing material may be used to reinforce the composite implantof the invention. In addition, biodegradable textile scaffolds, such as,for example, meshes, of the same structure and chemistry or differentstructures and chemistries can be overlaid on top of one another tofabricate biocompatible tissue implants with superior mechanicalstrength.

The source of viable tissue can also vary, and the tissue can have avariety of configurations. In one embodiment, however, the tissue is inthe form of finely minced tissue fragments, which enhance theeffectiveness of the regrowth and healing response. In anotherembodiment, the viable tissue can be in the form of a tissue slice orstrip that harvested from healthy tissue that contains viable cellscapable of tissue regeneration and/or remodeling, as described in U.S.patent application Ser. No. 10/729,046 filed Dec. 5, 2003 and entitled“Viable Tissue Repair Implants and Methods of Use.” The tissue slice ispreferably harvested to have a geometry that is suitable forimplantation at the site of the injury or defect, and the harvestedtissue slice is preferably dimensioned to allow the viable cellscontained within the tissue slice to migrate out and proliferate andintegrate with tissue surrounding the repair site.

Suitable tissue that can be used to obtain viable tissue includes, forexample, cartilage tissue, meniscal tissue, ligament tissue, tendontissue, skin tissue, bone tissue, muscle tissue, periosteal tissue,pericardial tissue, synovial tissue, nerve tissue, fat tissue, kidneytissue, bone marrow, liver tissue, bladder tissue, pancreas tissue,spleen tissue, intervertebral disc tissue, embryonic tissue, periodontaltissue, vascular tissue, blood, and combinations thereof. The tissueused to construct the tissue implant can be autogeneic tissue,allogeneic tissue, or xenogeneic tissue.

The particle size of each tissue fragment can also vary. By way ofnon-limiting example, the tissue size can be in the range of about 0.1and 3 mm³, in the range of about 0.5 and 1 mm³, in the range of about 1to 2 mm³, or in the range of about 2 to 3 mm³, but preferably the tissueparticle is less than 1 mm³.

The viable tissue can also optionally be combined with a variety ofother materials, including carriers, such as a gel-like carrier or anadhesive. By way of non-limiting example, the gel-like carrier can be abiological or synthetic hydrogel such as hyaluronic acid, fibrin glue,fibrin clot, collagen gel, collagen-based adhesive, alginate gel,crosslinked alginate, chitosan, synthetic acrylate-based gels, plateletrich plasma (PRP), platelet poor plasma (PPP), PRP clot, PPP clot,blood, blood clot, blood component, blood component clot, Matrigel,agarose, chitin, chitosan, polysaccharides, poly(oxyalkylene), acopolymer of poly(ethylene oxide)-poly(propylene oxide), poly(vinylalcohol), laminin, elasti, proteoglycans, solubilized basement membrane,or combinations thereof. Suitable adhesives include, but are not limitedto, hyaluronic acid, fibrin glue, fibrin clot, collagen gel,collagen-based adhesive, alginate gel, crosslinked alginate,gelatin-resorcin-formalin-based adhesive, mussel-based adhesive,dihydroxyphenylalanine (DOPA)-based adhesive, chitosan,transglutaminase, poly(amino acid)-based adhesive, cellulose-basedadhesive, polysaccharide-based adhesive, synthetic acrylate-basedadhesives, platelet rich plasma (PRP), platelet poor plasma (PPP), PRPclot, PPP clot, blood, blood clot, blood component, blood componentclot, polyethylene glycol-based adhesive, Matrigel, MonostearoylGlycerol co-Succinate (MGSA), Monostearoyl Glycerolco-Succinate/polyethylene glycol (MGSA/PEG) copolymers, laminin,elastin, proteoglycans, and combinations thereof.

The viable tissue can also be contacted with a matrix-digesting enzymeto facilitate tissue migration out of the extracellular matrixsurrounding the viable tissue. The enzymes can be used to increase therate of cell migration out of the extracellular matrix and into thetissue defect or injury, or scaffold material. Suitable matrix-digestingenzymes that can be used in the present invention include, but are notlimited to, collagenase, chondroitinase, trypsin, elastase,hyaluronidase, peptidase, thermolysin, matrix metalloproteinase,gelatinase and protease. Preferably, the concentration of minced tissueparticles in the gel-carrier is in the range of approximately 1 to 1000mg/cm3, and more preferably in the range of about 1 to 200 mg/cm3.

Other viable tissue sources and methods for preparing viable tissues aredisclosed in U.S. patent application Ser. No. 10/723,982 entitled“Conformable Tissue Repair Implant Capable Of Injection Delivery,” filedon Nov. 26, 2003 and incorporated by referenced herein in its entirety.

In use, the composite implant is preferably prepared by obtaining aviable tissue, preparing the tissue if necessary, and loading the tissueinto the pocket(s) in the tissue scaffold. The tissue used to obtainviable tissue sample can vary. In an exemplary embodiment, however, theviable tissue is obtained from or includes finely minced tissuefragments which enhance the effectiveness of the regrowth and healingresponse. The minced tissue fragments can be obtained using any of avariety of conventional techniques, such as for example, by biopsy orother surgical removal. Preferably, the tissue sample is obtained duringthe repair surgery to minimize the total number of surgeries performedon the patient. Once a sample of living tissue has been obtained, thesample can then be processed under sterile conditions to create asuspension having at least one minced, or finely divided tissueparticle. A carrier, such as a gel-like carrier or an adhesive, canoptionally be used to form the suspension. It is also possible toharvest the tissue in minced form such that further processing is notnecessary.

Once the viable tissue is prepared, the scaffold is loaded, andoptionally sealed if necessary. The composite can then be implanted andretained at the defect site by the force of compression against thetissue implant by the surrounding tissue. For instance, the tissueimplant can be dimensioned to have a slightly larger overall size thanthe area of the defect so that, upon implantation, the tissue implantcan form a tight, interference fit within the defect. Alternatively, thetissue implant can be secured to the defect using any conventionalmethod such as with a retaining element or an adhesive. The retainingelement can be, for example, a fastener, staple, tissue tack, suture,adhesive, or any combination of these. One skilled in the art willappreciate that a variety of techniques can be used to attach the tissueimplant to the surrounding tissue.

In another embodiment of the present invention, a bioactive agent may beincorporated within and/or applied to the tissue scaffolds, and/or itcan be applied to the viable tissue. Preferably, the bioactive agent isincorporated within, or coated on, the scaffold prior to the addition ofviable tissue to the scaffold. The bioactive agent(s) can be selectedfrom among a variety of effectors that, when present at the site ofinjury, promote healing and/or regeneration of the affected tissue. Inaddition to being compounds or agents that actually promote or expeditehealing, the effectors may also include compounds or agents that preventinfection (e.g., antimicrobial agents and antibiotics), compounds oragents that reduce inflammation (e.g., anti-inflammatory agents),compounds that prevent or minimize adhesion formation, such as oxidizedregenerated cellulose (e.g., INTERCEED® and SURGICEL®, available fromEthicon, Inc.), hyaluronic acid, and compounds or agents that suppressthe immune system (e.g., immunosuppressants).

By way of non-limiting example, other types of effectors present withinthe implant of the present invention can include heterologous orautologous growth factors, proteins (including matrix proteins),peptides, antibodies, enzymes, platelets, platelet rich plasma,glycoproteins, hormones, cytokines, glycosaminoglycans, nucleic acids,analgesics, viruses, virus particles, and cell types. It is understoodthat one or more effectors of the same or different functionality may beincorporated within the implant.

Examples of suitable effectors include the multitude of heterologous orautologous growth factors known to promote healing and/or regenerationof injured or damaged tissue. These growth factors can be incorporateddirectly into the scaffold, or alternatively, the scaffold can include asource of growth factors, such as for example, platelets. “Bioactiveagents,” as used herein, can include one or more of the following:chemotactic agents; therapeutic agents (e.g., antibiotics, steroidal andnon-steroidal analgesics and anti-inflammatories, anti-rejection agentssuch as immunosuppressants and anti-cancer drugs); various proteins(e.g., short term peptides, bone morphogenic proteins, glycoprotein andlipoprotein); cell attachment mediators; biologically active ligands;integrin binding sequence; ligands; various growth and/ordifferentiation agents and fragments thereof (e.g., epidermal growthfactor (EGF), hepatocyte growth factor (HGF), vascular endothelialgrowth factors (VEGF), fibroblast growth factors (e.g., bFGF), plateletderived growth factors (PDGF), insulin derived growth factor (e.g.,IGF-1, IGF-II) and transforming growth factors (e.g., TGF-β I-III),parathyroid hormone, parathyroid hormone related peptide, bonemorphogenic proteins (e.g., BMP-2, BMP-4; BMP-6; BMP-12), sonichedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8),recombinant human growth factors (e.g., MP52), cartilage-derivedmorphogenic proteins (CDMP-1)); small molecules that affect theupregulation of specific growth factors; tenascin-C; hyaluronic acid;chondroitin sulfate; fibronectin; decorin; thromboelastin;thrombin-derived peptides; heparin-binding domains; heparin; heparansulfate; DNA fragments and DNA plasmids. Suitable effectors likewiseinclude the agonists and antagonists of the agents described above. Thegrowth factor can also include combinations of the growth factorsdescribed above. In addition, the growth factor can be autologous growthfactor that is supplied by platelets in the blood. In this case, thegrowth factor from platelets will be an undefined cocktail of variousgrowth factors. If other such substances have therapeutic value in theorthopaedic field, it is anticipated that at least some of thesesubstances will have use in the present invention, and such substancesshould be included in the meaning of “bioactive agent” and “bioactiveagents” unless expressly limited otherwise.

Biologically derived agents, suitable for use as effectors, include oneor more of the following: bone (autograft, allograft, and xenograft) andderivates of bone; cartilage (autograft, allograft and xenograft),including, for example, meniscal tissue, and derivatives; ligament(autograft, allograft and xenograft) and derivatives; derivatives ofintestinal tissue (autograft, allograft and xenograft), including forexample submucosa; derivatives of stomach tissue (autograft, allograftand xenograft), including for example submucosa; derivatives of bladdertissue (autograft, allograft and xenograft), including for examplesubmucosa; derivatives of alimentary tissue (autograft, allograft andxenograft), including for example submucosa; derivatives of respiratorytissue (autograft, allograft and xenograft), including for examplesubmucosa; derivatives of genital tissue (autograft, allograft andxenograft), including for example submucosa; derivatives of liver tissue(autograft, allograft and xenograft), including for example liverbasement membrane; derivatives of skin tissue; platelet rich plasma(PRP), platelet poor plasma, bone marrow aspirate, demineralized bonematrix, insulin derived growth factor, whole blood, fibrin and bloodclot. Purified ECM and other collagen sources are also appropriatebiologically derived agents. If other such substances have therapeuticvalue in the orthopaedic field, it is anticipated that at least some ofthese substances will have use in the present invention, and suchsubstances should be included in the meaning of “biologically derivedagent” and “biologically derived agents” unless expressly limitedotherwise.

Biologically derived agents also include bioremodelable collageneoustissue matrices. The terms “bioremodelable collageneous tissue matrix”and “naturally occurring bioremodelable collageneous tissue matrix”include matrices derived from native tissue selected from the groupconsisting of skin, artery, vein, pericardium, heart valve, dura mater,ligament, bone, cartilage, bladder, liver, stomach, fascia andintestine, whatever the source. Although the term “naturally occurringbioremodelable collageneous tissue matrix” is intended to refer tomatrix material that has been cleaned, processed, sterilized, andoptionally crosslinked, it is not within the definition of a naturallyoccurring bioremodelable collageneous tissue matrix to purify thenatural fibers and reform a matrix material from purified naturalfibers.

The proteins that may be present within the implant include proteinsthat are secreted from a cell or other biological source, such as forexample, a platelet, which is housed within the implant, as well asthose that are present within the implant in an isolated form. Theisolated form of a protein typically is one that is about 55% or greaterin purity, i.e., isolated from other cellular proteins, molecules,debris, etc. More preferably, the isolated protein is one that is atleast 65% pure, and most preferably one that is at least about 75 to 95%pure. Notwithstanding the above, one of ordinary skill in the art willappreciate that proteins having a purity below about 55% are stillconsidered to be within the scope of this invention. As used herein, theterm “protein” embraces glycoproteins, lipoproteins, proteoglycans,peptides, and fragments thereof. Examples of proteins useful aseffectors include, but are not limited to, pleiotrophin, endothelin,tenascin, fibronectin, fibrinogen, vitronectin, V-CAM, I-CAM, N-CAM,selectin, cadherin, integrin, laminin, actin, myosin, collagen,microfilament, intermediate filament, antibody, elastin, fibrillin, andfragments thereof.

Glycosaminoglycans, highly charged polysaccharides which play a role incellular adhesion, may also serve as effectors according to the presentinvention. Exemplary glycosaminoglycans useful as effectors include, butare not limited to, heparan sulfate, heparin, chondroitin sulfate,dermatan sulfate, keratan sulfate, hyaluronan (also known as hyaluronicacid), and combinations thereof.

The tissue scaffolds of the present invention can also have cellsincorporated therein. Suitable cell types that can serve as effectorsaccording to this invention include, but are not limited to, osteocytes,osteoblasts, osteoclasts, fibroblasts, stem cells, pluripotent cells,chondrocyte progenitors, chondrocytes, endothelial cells, macrophages,leukocytes, adipocytes, monocytes, plasma cells, mast cells, umbilicalcord cells, stromal cells, mesenchymal stem cells, epithelial cells,myoblasts, tenocytes, ligament fibroblasts, neurons, bone marrow cells,synoviocytes, embryonic stem cells; precursor cells derived from adiposetissue; peripheral blood progenitor cells; stem cells isolated fromadult tissue; genetically transformed cells; a combination ofchondrocytes and other cells; a combination of osteocytes and othercells; a combination of synoviocytes and other cells; a combination ofbone marrow cells and other cells; a combination of mesenchymal cellsand other cells; a combination of stromal cells and other cells; acombination of stem cells and other cells; a combination of embryonicstem cells and other cells; a combination of precursor cells isolatedfrom adult tissue and other cells; a combination of peripheral bloodprogenitor cells and other cells; a combination of stem cells isolatedfrom adult tissue and other cells; and a combination of geneticallytransformed cells and other cells. If other cells are found to havetherapeutic value in the orthopaedic field, it is anticipated that atleast some of these cells will have use in the present invention, andsuch cells should be included within the meaning of “cell” and “cells”unless expressly limited.

Cells typically have at their surface receptor molecules which areresponsive to a cognate ligand (e.g., a stimulator). A stimulator is aligand which when in contact with its cognate receptor induce the cellpossessing the receptor to produce a specific biological action. Forexample, in response to a stimulator (or ligand) a cell may producesignificant levels of secondary messengers, like Ca+2, which then willhave subsequent effects upon cellular processes such as thephosphorylation of proteins, such as (keeping with our example) proteinkinase C. In some instances, once a cell is stimulated with the properstimulator, the cell secretes a cellular messenger usually in the formof a protein (including glycoproteins, proteoglycans, and lipoproteins).This cellular messenger can be an antibody (e.g., secreted from plasmacells), a hormone, (e.g., a paracrine, autocrine, or exocrine hormone),a cytokine, or natural or synthetic fragments thereof.

The tissue implants of the invention can also be used in gene therapytechniques in which nucleic acids, viruses, or virus particles deliver agene of interest, which encodes at least one gene product of interest,to specific cells or cell types. Accordingly, the biological effectorcan be a nucleic acid (e.g., DNA, RNA, or an oligonucleotide), a virus,a virus particle, or a non-viral vector. The viruses and virus particlesmay be, or may be derived from, DNA or RNA viruses. The gene product ofinterest is preferably selected from the group consisting of proteins,polypeptides, interference ribonucleic acids (iRNA) and combinationsthereof.

Once the applicable nucleic acids and/or viral agents (i.e., viruses orviral particles) are incorporated into the biocompatible scaffold of thetissue repair implant, the implant can then be implanted into aparticular site to elicit a type of biological response. The nucleicacid or viral agent can then be taken up by the cells and any proteinsthat they encode can be produced locally by the cells. In oneembodiment, the nucleic acid or viral agent can be taken up by the cellswithin the tissue fragment of the minced tissue suspension, or, in analternative embodiment, the nucleic acid or viral agent can be taken upby the cells in the tissue surrounding the site of the injured tissue.One skilled in the art will recognize that the protein produced can be aprotein of the type noted above, or a similar protein that facilitatesan enhanced capacity of the tissue to heal an injury or a disease,combat an infection, or reduce an inflammatory response. Nucleic acidscan also be used to block the expression of unwanted gene product thatmay impact negatively on a tissue repair process or other normalbiological processes. DNA, RNA and viral agents are often used toaccomplish such an expression blocking function, which is also known asgene expression knock out.

One of ordinary skill in the art will appreciate that the identity ofthe bioactive agent may be determined by a surgeon, based on principlesof medical science and the applicable treatment objectives. It isunderstood that the bioactive agent or effector of the tissue repairimplant can be incorporated within the tissue scaffold before or aftermanufacture of the tissue scaffold, or before or after the surgicalplacement of the implant.

Prior to surgical placement, the tissue scaffold can be placed in asuitable container comprising the bioactive agent. After an appropriatetime and under suitable conditions, the scaffold will become impregnatedwith the bioactive agent. Alternatively, the bioactive agent can beincorporated within the scaffold by, for example, using an appropriatelygauged syringe to inject the biological agent(s) into the scaffold.Other methods well known to those of skilled in the art can be appliedin order to load a scaffold with an appropriate bioactive agent, such asmixing, pressing, spreading, centrifuging and placing the bioactiveagent into the scaffold. Alternatively, the bioactive agent can be mixedwith a gel-like carrier prior to injection into the scaffold.

Following surgical placement, an implant wherein the biocompatiblescaffold is devoid of any bioactive agent can be infused with biologicalagent(s), or an implant wherein the scaffold includes at least onebioactive agent can be augmented with a supplemental quantity of thebioactive agent. One method of incorporating a bioactive agent within asurgically installed implant is by injection using an appropriatelygauged syringe.

The amount of the bioactive agent included with a biocompatible scaffoldwill vary depending on a variety of factors, including the size of thescaffold, the material from which the scaffold is made, the porosity ofthe scaffold, the identity of the biologically component, and theintended purpose of the tissue repair implant. One skilled in the artcan readily determine the appropriate quantity of bioactive agent toinclude within a biocompatible scaffold for a given application in orderto facilitate and/or expedite the healing of tissue. The amount ofbioactive agent will, of course, vary depending upon the identity of thebioactive agent and the given application.

The following non-limiting examples are illustrative of the principlesand practice of this invention. Numerous additional embodiments withinthe scope and spirit of the invention will become apparent to thoseskilled in the art.

EXAMPLE 1

Cellular migration and new matrix formation of bovine minced meniscaltissue (BMT) into a small intestine submucosa (SIS) tissue scaffold wasevaluated and compared. Meniscal tissue was harvested from the white andred-white zone of an adult bovine menisci, and the tissue was minced toform a viable tissue source. A scaffold was prepared from a smallintestine submucosa (SIS), and a slit was made in the scaffold to form apocket. The minced tissue was loaded at 20 mg/cm2 into the SIS scaffoldthrough the slit to form a composite implant. The composite implant wasplaced in a pocket created in the hemithorax through one skin incisionof a mouse. Tacking sutures of 5-0 Ethibond Excel® were used to tack theskin to musculature around each composite implant to preventsubcutaneous migration.

After 4 weeks, the composite implant was prepared for histologicalevaluation by fixing the implant in 10% buffered formalin, sectioningthe implant to form samples, and staining the samples withHematoxylin/Eosin (H/E). A photomicrograph of the composite implant isshown in FIG. 8B, which can be compared to a photomicrograph of acontrol sample shown in FIG. 8A. The control was prepared using the sameaforementioned procedure, however minced tissue was not loaded into thescaffold.

FIG. 8A shows the control tissue scaffold 80 a having no native tissuecells. FIG. 8B, on the other hand, shows native meniscal tissue cells 84b that have migrated into the implant and that are present between theminced BMT 82 b, and it also shows the minced BMT 82 b being remodeledby the native meniscal tissue cells 84 b.

EXAMPLE 2

Cellular migration and new matrix formation of bovine minced meniscaltissue (BMT) into a small intestine submucosa (SIS) tissue scaffold wasevaluated and compared. Meniscal tissue was harvested from the white andred-white zone of adult bovine menisci, and the tissue was minced toform a viable tissue source. A scaffold was prepared from a smallintestine submucosa (SIS), and a slit was made in the scaffold to form apocket. The minced tissue was combined with platelet rich plasma (PRP),and the composition was loaded into the SIS scaffold at 20 mg/cm2through the slit to form a composite implant. The composite implant wasplaced in a pocket created in the hemithorax through one skin incisionof a mouse. Tacking sutures of 5-0 Ethibond Excel® were used to tack theskin to musculature around each composite implant to preventsubcutaneous migration.

After 4 weeks, the composite implant was prepared for histologicalevaluation by fixing the implant in 10% buffered formalin, sectioningthe implant to form samples, and staining the samples withHematoxylin/Eosin (H/E). A photomicrograph of the composite implant isshown in FIG. 8C. Reference number 82 c shows the minced BMT and PRPbeing remolded by native meniscal tissue cells 84 c that have migratedinto the implant.

EXAMPLE 3

Cellular migration and new matrix formation of bovine minced meniscaltissue (BMT) into a small intestine submucosa (SIS) tissue scaffold wasevaluated and compared. Meniscal tissue was harvested from the white andred-white zone of adult bovine menisci, and the tissue was minced. PRPwas added to the minced BMT. A viable tissue source was then prepared bycombining the BMT and PRP with a bioresorbable polymer scaffold(referred to as “FPV”) in a 50:50 ratio. The resorbable scaffold usedwas a lyophilized foam scaffold (65% polyglycolic acid/35%Polycaprolactone) reinforced with nonwoven fibers (mixture ofPDS[Polydioxanone] and Vicryl). A scaffold was prepared from a smallintestine submucosa (SIS), and a slit was made in the scaffold to form apocket. The viable tissue source of FPV, BMT, and PRP was loaded at 20mg/cm2 into the SIS scaffold through the slit to form a compositeimplant. The composite implant was placed in a pocket created in thehemithorax through one skin incision of a mouse. Tacking sutures of 5-0Ethibond Excel® were used to tack the skin to musculature around eachcomposite implant to prevent subcutaneous migration.

After 4 weeks, the composite implant was prepared for histologicalevaluation by fixing the implant in 10% buffered formalin, sectioningthe implant to form samples, and staining the samples withHematoxylin/Eosin (H/E). A photomicrograph of the composite implant isshown in FIG. 8D, which shows a significant amount of native meniscaltissue cell 84 d migration into FPV scaffold 80 d and around and betweenthe BMT, and PRP tissue source 82 d.

EXAMPLE 4

Cellular migration and new matrix formation of bovine cartilage tissue(BCT) into a small intestine submucosa (SIS) tissue scaffold wasevaluated and compared. Cartilage tissue was harvested from the femoralcondyles of adult bovine animals, and the tissue was minced. PRP wasadded to the minced BCT. A viable tissue source was then prepared bycombining the BCT and PRP with a bioresorbable polymer scaffold(referred to as “PV”) in a 50:50 ratio. The resorbable scaffold used wasa nonwoven scaffold (mixture of PDS[Polydioxanone] and Vicryl). Ascaffold was prepared from a small intestine submucosa (SIS), and a slitwas made in the scaffold to form a pocket. The viable tissue source ofPV, BCT, and PRP was loaded at 20 mg/cm2 into the SIS scaffold throughthe slit to form a composite implant. The composite implant was placedin a pocket created in the hemithorax through one skin incision of amouse. Tacking sutures of 5-0 Ethibond Excel® were used to tack the skinto musculature around each composite implant to prevent subcutaneousmigration.

After 4 weeks, the composite implant was prepared for histologicalevaluation by fixing the implant in 10% buffered formalin, sectioningthe implant to form samples, and staining the samples withHematoxylin/Eosin (H/E). A photomicrograph of the composite implant isshown in FIG. 8E. Some native cartilage tissue cell 84 e migration intothe tissue scaffold 80 e and around and between the viable tissue source82 e is shown.

EXAMPLE 5

The effect of using a bioactive substance on native meniscal cellmigration into a tissue scaffold was evaluated. Menisci were harvestedfrom bovine knees and 4 mm explants were taken from the white andred/white regions. A 2 mm punch biopsy was removed from the center ofthe explants prior to scaffold insertion. Bioresorbable foam scaffolds(60% Polylactic acid and 40% Polycaprolactone), 2 mm in diameter, weretreated either with blood, Platelet Rich Plasma (PRP), or 8 timesconcentrated PRP, and they were inserted in the center of the explants.The explants with scaffolds were cultured for 2 and 3 weeks understandard cell culture conditions with changes in media occurring everyother day. At 2 and 3 weeks, the scaffolds within the explants wereremoved and cell number was estimated by quantitation of DNA using theCyQuant assay.

FIG. 9A illustrates the advantages of using a bioactive agent, and inparticular it demonstrates that native meniscal cell migration into thescaffold is significantly improved with the use of a bioactive agent,such as a platelet rich plasma (PRP). As shown, the amount of nativetissue cells that migrated into the implant after two and three weekssignificantly increases in the implants containing PRP as compared tothe control and the implant containing blood.

EXAMPLE 6

The effect of using a bioactive substance on native cell migration intoa tissue scaffold was evaluated. Menisci were harvested from bovineknees and 4 mm explants were taken from the white and red/white regions.A 2 mm punch biopsy was removed from the center of the explants prior toscaffold insertion. Bioresorbable foam scaffolds (65% Polyglycolic acidand 35% Polycaprolactone), reinforced with PDS, (Polydioxanone) mesh, 2mm in diameter, were soaked in 100 μl of CDMP-1 at the concentrations of10 ng/ml, 100 ng/ml and 300 ng/ml. Scaffolds were lyophilized overnight,such that the growth factor was lyophilized in the scaffold and theninserted in the center of the explants The explants with scaffolds werecultured for 3 weeks under standard cell culture conditions with changesin media occurring every other day. At 2 and 3 weeks, the scaffoldswithin the explants were removed and cell number estimated byquantitation of DNA using the CyQuant assay.

FIG. 9B demonstrates the amount of native meniscal tissue cells thatmigrate into an implant formed from 65% polyglycolic acid/35%polycaprolactone reinforced with a PDS® mesh after 14 days and after 21days. As shown, the implant containing no CDMP-1 and the implantcontaining only 10 ng/ml of CDMP-1 did not receive as much native tissuecells after 14 and 21 days as the implants containing 100 ng/ml ofCDMP-1 and 300 ng/ml of CDMP-1, which showed a significant improvementin native meniscal tissue cell migration.

EXAMPLE 7

The effect of using a bioactive substance on native cell migration intoa tissue scaffold was evaluated. Menisci were harvested from bovineknees and 4 mm explants were taken from the white and red/white regions.A 2 mm punch biopsy was removed from the center of the explants prior toscaffold insertion. Bioresorbable foam scaffolds (65% Polyglycolic acidand 35% Polycaprolactone), reinforced with PDS, (Polydioxanone) mesh, 2mm in diameter, were soaked in 100 μl of CDMP-1 at the concentrations of150 ng/ml. Scaffolds were lyophilized overnight, such that the growthfactor was lyophilized in the scaffold and then inserted in the centerof the explants The explants with scaffolds were cultured for 3 weeksunder standard cell culture conditions with changes in media occurringevery other day. At 3 weeks, the explants were removed and processedhistology for staining with Hematoxylin/Eosin (H/E).

FIGS. 10A and 10B show a photomicrograph of the explants. FIG. 10A is aphotomicrograph of the first explant which was untreated, serving as thecontrol, and FIG. 10B is a photomicrograph of the meniscal explant withCDMP-1. As shown, the explant in FIG. 10B contains a significant amountof native tissue cell as compared to the implant in FIG. 10A.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

1-41. (canceled)
 42. A medical method, comprising: mincing viable tissuecomprising naturally occurring cells and their extracellular matrix toform finely minced tissue fragments and loading the finely minced tissuefragments into a tissue scaffold; and implanting the tissue scaffoldwith the finely minced tissue fragments disposed therein at a defectsite in a patient's body such that native tissue surrounding the tissuescaffold abuts the opening in the pocket to maintain the finely mincedtissue fragments therein.
 43. The method of claim 42, further comprisingthe step of applying at least one bioactive substance to the tissuefragments to stimulate cell growth.
 44. The method of claim 43, whereinthe bioactive substance is selected from the group consisting of a bloodclots, platelet rich plasma, cartilage-derived morphogenic proteins,recombinant human growth factors, and combinations thereof.
 45. Themethod of claim 42, wherein the tissue scaffold is substantiallywedge-shaped; the finely minced tissue fragments are loaded into apocket of a tissue scaffold; the pocket is the only pocket formed in thetissue scaffold; and the pocket comprises a hollow interior formed inthe tissue scaffold.
 46. The method of claim 42, wherein the tissuescaffold is substantially wedge-shaped; the finely minced tissuefragments are loaded into a pocket of a tissue scaffold; the pocket isthe only pocket formed in the tissue scaffold; and the pocket is onelumen extending into the tissue scaffold.
 47. The method of claim 42,wherein the tissue scaffold includes an upper layer and a lower layer;and the finely minced tissue fragments are loaded into the tissuescaffold so as to be sandwiched between the upper layer and the lowerlayer.
 48. The method of claim 47, further comprising, after loading thefinely minced tissue fragments into the tissue scaffold and before theimplanting of the tissue scaffold, sealing the upper layer and the lowerlayer around an entire perimeter thereof.
 49. The method of claim 47,wherein the tissue scaffold is implanted at the defect site without theupper layer and the lower layer being sealed together.
 50. The method ofclaim 42, wherein the tissue fragment has a thickness in the range fromabout 200 um to about than 3 mm; and the tissue fragment has a particlesize in the range from about 0.5 mm³ to about 3 mm³.
 51. The method ofclaim 42, wherein the viable tissue is one of meniscal tissue andcartilage tissue.
 52. The method of claim 42, wherein cells from theviable tissue in the pocket of the scaffold populate at least a portionof the scaffold.
 53. The method of claim 42, wherein at least a portionof the scaffold is populated with cells from the native tissue followingimplantation.
 54. The method of claim 42, wherein the tissue scaffold isformed only of at least one natural polymer.
 55. The method of claim 54,wherein the at least one natural polymer includes at least one of afibrin-based material, a collagen-based material, a hyaluronicacid-based material, a glycoprotein-based material, a cellulose-basedmaterial, and silk.
 56. The method of claim 42, wherein the tissuescaffold is formed from a bioresorbable, synthetic polymeric material.57. A medical device, comprising: a porous tissue scaffold including anupper layer and a lower layer that is sealed to the upper layer; andfinely minced fragments of viable tissue comprising naturally occurringcells and their extracellular matrix, the naturally occurring cells andtheir extracellular matrix being native to the viable tissue; whereinthe finely minced fragments of viable tissue are loaded in a centralopen area of the tissue scaffold located between the upper layer and thelower layer.
 58. The device of claim 57, wherein the upper layer and thelower layer are sealed together around an entire perimeter thereof. 59.The device of claim 57, wherein the tissue scaffold is substantiallywedge-shaped; the tissue scaffold has a first side, a second sideopposed to the first side, a top, and a bottom opposed to the top; andthe upper layer and the lower layer are sealed together at an end of thetissue scaffold so as to connect the top, the bottom, the first side,and the second side of the tissue scaffold.
 60. The device of claim 57,wherein the viable tissue is viable cartilage tissue.
 61. The device ofclaim 57, wherein the viable tissue is viable meniscal tissue.